【摘要】：研究背景線粒體病(mitochondrial diseases)一組由線粒體DNA (mtDNA)或核DNA(nDNA)缺陷導致的線粒體結(jié)構(gòu)和功能障礙、ATP合成不足所致的多系統(tǒng)疾病,包括：原發(fā)性線粒體病,由編碼線粒體功能相關蛋白的基因突變引起的；繼發(fā)性線粒體病,由藥物、環(huán)境、感染等引起。該病于1959年被首次報道,此后幾十年越來越多的疾病發(fā)現(xiàn)與線粒體功能異常有關,這其中不僅包括基因突變導致線粒體病,也包括一些常見疾病,例如糖尿病、腫瘤、肥胖、阿爾茲海默病、帕金森病、亨廷頓病和原發(fā)性癲癇等。Holt于1988年首次在線粒體肌病患者中發(fā)現(xiàn)mtDNA缺失,28年來,通過對線粒體患者及其家系的研究,幾百種致病性核基因和線粒體基因被發(fā)現(xiàn),從而證實了核基因和線粒體基因突變是原發(fā)性線粒體病的重要遺傳因素。如mtDNA點突變可以導致線粒體腦肌病伴高乳酸血癥、卒中樣發(fā)作(MELAS),mtDNA單一大片段缺失引起眼外肌麻痹(CPEO),核基因Twinkle的致病突變可以導致家族性的眼外肌麻痹等。隨著測序技術(shù)的發(fā)展,盡管越來越多的致病病基因被發(fā)現(xiàn)和報道,但是依然有很多未解的問題：①線粒體病基因型和表型多樣性,臨床復雜多樣,受累器官不同,各年齡均可發(fā)病,這對于線粒體的診斷是個難題。②同一種致病性突變可以導致截然不同的臨床表現(xiàn),特殊的線粒體病亞型可以表現(xiàn)為特異性組織損害；③線粒體病的治療,目前對于預防發(fā)作和延緩進展的治療還是非常有限。自從成纖維細胞生長因子21在2005年被發(fā)現(xiàn)以來,由于其在機體能量代謝調(diào)控方面的廣泛作用,引起了國內(nèi)外研究團隊的廣泛興趣。FGF21作為成纖維細胞生長因子(FGF)家族中特殊的一員,具有許多內(nèi)分泌功能。FGF21和其他成纖維細胞因子家族不同,FGF21可以被分泌到血液中,作用于全身各個系統(tǒng)。人類的FGF21由187個氨基酸組成,主要由肝臟分泌,調(diào)節(jié)機體的葡萄糖和脂肪的代謝。研究發(fā)現(xiàn)：正常情況下,血液中的FGF21的濃度很低,在饑餓和酮體飲食的情況下,血中FGF21的濃度明顯升高。此外,二型糖尿病、冠心病、肝功能異常等疾病都可以導致血FGF21輕度的升高。近期的研究發(fā)現(xiàn),在線粒體病的小鼠模型以及線粒體病患者中,血清FGF21水平升高。雖然FGF21因子的作用在許多病理過程中被報道和研究,但是它們在線粒體病中的作用卻一直未被闡明,此外關于FGF21如何調(diào)節(jié)細胞線粒體的功能的研究也很少。線粒體是細胞內(nèi)三大物質(zhì)代謝的主要場所,因此線粒體的功能缺陷可以導致糖、脂肪酸和氨基酸代謝的異常,不僅表現(xiàn)為血乳酸、丙酮酸和丙氨酸的異常,也可以引起尿液中物質(zhì)代謝中間產(chǎn)物的升高,如2-氧戊二酸鹽、琥珀酸鹽、延胡索酸鹽、蘋果酸鹽等。線粒體功能異常情況下,機體通過正反饋和負反饋機制對物質(zhì)和能量代謝狀態(tài)進行重新調(diào)控,最大限度地對線粒體的功能缺陷進行代償,保證細胞相對正常的生理狀態(tài)。這種能量代謝的調(diào)控機制被稱為線粒體能量代謝重構(gòu)。線粒體功能缺陷引起的能量代謝重構(gòu)主要依賴于以線粒體為中心的生物能學體系。細胞內(nèi)ATP、acetyl-CoA、NAD+、SAM的水平可直接影響DNA的甲基化、組蛋白的乙�；约暗鞍椎牧姿峄鸵阴；�,并通過上述因子重新調(diào)控細胞的能量代謝過程。為此,本課題圍繞線粒體病基因型—臨床表型異質(zhì)性和能量代償調(diào)控機制進行了以下幾方面的工作：第一部分線粒體病中FGF21介導的能量代謝重構(gòu)代償線粒體功能缺陷的機制研究研究目的本部分圍繞線粒體病中FGF21細胞因子如何重構(gòu)整個機體的能量代謝過程,采用分子干預和體外實驗研究的手段,明確FGF21在以肌肉損害為主的線粒體病患者肌肉標本中是否升高,闡明FGF21對機體能量代謝的影響,探索線粒體病基因型—臨床表現(xiàn)關系復雜多變的機制。材料方法1)利用SYBR GREEN實時定量PCR方法檢測分析線粒體病患者肌肉組織內(nèi)FGF21 mRNA的表達,蛋白免疫電泳的方法分析線粒體病肌肉組織中FGF21蛋白的表達。2)利用線粒體呼吸鏈抑制劑,如魚藤酮、寡霉素,以及解偶聯(lián)劑FCCP誘導成肌細胞線粒體的功能障礙3)利用SYBR GREEN實時定量PCR方法檢測分析FGF21誘導前后,肌細胞物質(zhì)代謝相關基因的表達情況4)采用mtDNA數(shù)量、細胞內(nèi)ATP含量、檸檬酸合酶活性以及線粒體ROS水平等方法評估成肌細胞的線粒體功能5)海馬細胞能量分析儀測量細胞氧耗量和細胞外酸化率,獲得細胞基礎呼吸率、ATP產(chǎn)生量、質(zhì)子漏、最大呼吸率、細胞基礎糖酵解率以及最大糖酵解能力等指標。6)通過分析成肌細胞最大呼吸率評估細胞抵抗ROS的能力。7)蛋白免疫組化分析FGF21處理后,成肌細胞內(nèi)mTOR-PGC1 α等信號通路的變化。8)雷帕霉素抑制mTOR蛋白激酶的活性,LY294002抑制PI3K-Akt通路。9)shRNA減少YY1和PGC1 α轉(zhuǎn)錄因子的表達,明確它們在FGF21促進能量代謝方面作用的分子機制。實驗結(jié)果線粒體腦肌病患者肌肉組織內(nèi)FGF21表達明顯升高,線粒體呼吸鏈抑制劑誘導的線粒體損傷能夠上調(diào)肌細胞FGF21的表達；FGF21因子能夠促進肌細胞線粒體代謝相關基因(CPT1A, CPT2, PPARy,IDH3A, GLUT1)的表達翻譯,增加成肌細胞mtDNA的數(shù)量,增強線粒體檸檬酸合酶的活性,增加細胞內(nèi)ATP的含量,增加成肌細胞的氧氣消耗率和促進細胞的糖酵解過程。FGF21促進成肌細胞能量代謝過程是通過激活細胞內(nèi)mTOR-YY 1-PGC1 α信號通路實現(xiàn)的,雷帕霉素、shRNA降低PGCl α的表達能夠減弱FGF21的這種促進作用。伴有線粒體功能障礙的一種組織可以通過分泌FGF21細胞因子重構(gòu)整個機體的能量代謝過程。血清中升高的FGF21細胞因子,一方面作用于肝臟導致酮體的產(chǎn)生；另一方面作用于脂肪組織,導致脂肪的分解；這些酮體和脂肪酸可以被肌肉、腦等其他器官組織攝取和利用。實驗結(jié)論①線粒體腦肌病患者肌肉組織內(nèi)FGF21表達明顯升高；②線粒體氧化呼吸功能異�？梢砸鸺〖毎麅�(nèi)FGF21細胞因子水平的升高,肌肉細胞通過分泌FGF21因子來代償線粒體能量代謝的異常；③肌肉組織通過分泌FGF21細胞因子,一方面作用于肌肉組織本身,激活細胞內(nèi)的mTOR-YY1-PGC1 α信號通路增強線粒體的功能和氧化呼吸效率,另一方面作用于肝臟和脂肪組織為肌肉供能；④伴有線粒體功能異常的肌肉組織可以通過分泌FGF21因子調(diào)控機體的能量代謝過程。第二部分線粒體基因C15620A多態(tài)位點影響G11778A突變臨床表型的研究研究目的線粒體基因G11778A突變是Leber遺傳性視神經(jīng)病(LHON)的常見致病突變位點,而G11778A突變引起Leigh綜合征(LS)確罕有人報道。我們報道一例攜帶有線粒體G11778A突變的LS患者的臨床和病理特點,并探討線粒體基因多態(tài)性位點C15620A對致病突變G11778A臨床表型的影響。材料方法采集一例臨床表現(xiàn)為LS的3歲漢族兒童相關病史、體格檢查以及其他輔助檢查資料,完善血、腦脊液乳酸、血尿有機酸、血脂酰肉堿和視神經(jīng)檢查,并行肌肉活檢和線粒體基因全長序列分析,以明確致病突變位點。分離培養(yǎng)患兒的皮膚成纖維細胞,對其進行線粒體呼吸鏈酶活性分析、細胞線粒體膜電位及細胞ATP含量檢測分析,以明確患兒的線粒體功能。實驗結(jié)果該患兒自幼發(fā)育較同齡兒慢,一歲八個月會走,兩歲會說話,在兩歲十個月的時候開始出現(xiàn)意向性震顫、右眼內(nèi)斜視和走路不穩(wěn)。神經(jīng)系統(tǒng)查體發(fā)現(xiàn)右眼內(nèi)斜視,走路不穩(wěn)和共濟失調(diào),眼底檢查未見明顯異常。顱腦MRI顯示雙側(cè)延髓、腦橋、中腦及小腦對稱性的長T1長T2信號,FLARI呈高信號。空腹血乳酸6.0mmol/L(正常值1.55mmol/L);腦脊液乳酸3. lmmol/L。肌肉切片酶組織化學染色提示部分肌纖維肌膜下琥珀酸脫氫酶活性增強。線粒體基因分析發(fā)現(xiàn)G11778A致病突變,其在肌肉組織突變率為91.6%,外周血的突變率為57%；同時并合并一個新的C15620A非致病突變,其在肌肉組織突變率分別為90%；患兒母親的外周血中也發(fā)現(xiàn)了這兩種突變,突變率分別是57%和79%。同時,患兒皮膚成纖維細胞呼吸鏈復合體Ⅰ活性、復合體Ⅲ酶活性和線粒體膜電位分別降低了67%、15%和50%。 C15620 A非致病突變可能通過降低復合體Ⅲ酶的活性而影響GI1778A致病突變的臨床表型。實驗結(jié)論線粒體基因GI1778A致病突變可以引起Leigh綜合征。線粒體基因C15620A多態(tài)位點可能過降低復合體Ⅲ酶的活性而影響致病性GI1778A突變的臨床表型,表現(xiàn)為Leigh綜合征。第三部分Twinkle基因突變導致的家族性眼外肌麻痹的兩例家系報道研究目的總結(jié)家族性眼外肌麻痹患者的肌肉病理、基因和臨床表現(xiàn)的特點,分析Twinkle基因突變引起眼外肌麻痹的肌肉病理表現(xiàn)和臨床特點。材料方法收集整理齊魯醫(yī)院神經(jīng)肌肉研究室2005年以來的兩例家族性眼外肌麻痹家系患者的臨床及其他輔助檢查資料,完善血乳酸、血脂酰肉堿及尿有機酸等檢查,總結(jié)肌肉活檢病理表現(xiàn),Twinkle基因測序分析明確致病位點。總結(jié)文獻資料,概括歸納Twinkle基因致病性突變的各種類型及其表型。實驗結(jié)果這兩個家系中的患者臨床表現(xiàn)相對較輕,僅表現(xiàn)為輕-中度的眼瞼下垂、輕度的眼外肌麻痹和進行性的肌無力。肌肉病理主要表現(xiàn)為少量的RRFs和COX酶活性缺失纖維。Twinkle基因測序分析發(fā)現(xiàn)了兩個已報道的致病性突變,分別是c.1423GC,p.A475P和c.1061GC,p.R354P。對兩個家系的先證者肌肉mtDNA分析發(fā)現(xiàn),它們都存在多發(fā)缺失突變。實驗結(jié)論Twinkle基因是家族性眼外肌麻痹的常見致病基因,p.A475P和p.R354P突變是常見的致病突變位點,它引起的臨床表現(xiàn)相對較輕,肌肉病理主要特點是少量散在分布的破碎樣紅纖維(RRFs)和COX酶活性缺失纖維。
[Abstract]:Background Mitochondrial diseases are a group of multisystem diseases caused by mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) deficiencies and ATP deficiency, including primary mitochondrial diseases, caused by mutations in genes encoding mitochondrial function-related proteins; secondary mitochondrial diseases, caused by deficiencies in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). Drugs, environment, infection, etc. The disease was first reported in 1959. Since then, more and more diseases have been found to be associated with mitochondrial dysfunction, including not only genetic mutations causing mitochondrial diseases, but also common diseases such as diabetes, cancer, obesity, Alzheimer's disease, Parkinson's disease, Huntington's disease and primary disease. In 1988, Holt first found mtDNA deletion in patients with mitochondrial myopathy. In the past 28 years, hundreds of pathogenic nuclear and mitochondrial genes have been found in patients with mitochondrial myopathy and their families, which confirms that nuclear and mitochondrial gene mutations are important genetic factors in primary mitochondrial diseases. Mitochondrial encephalomyopathy with hyperlactinemia, stroke-like seizures (MELAS), extraocular muscle paralysis (CPEO) caused by deletion of a single large fragment of mtDNA, and familial extraocular muscle paralysis caused by mutations in the nuclear gene Twinkle. With the development of sequencing technology, although more and more pathogenic genes have been found and reported, there are still some. Many unsolved problems are as follows: 1. Mitochondrial disease is characterized by genotypic and phenotypic diversity, clinical complexity, organ involvement, and age. This is a difficult problem for the diagnosis of mitochondrial disease. 2. The same pathogenic mutation can lead to distinct clinical manifestations, and special mitochondrial disease subtypes can manifest specific tissue damage. Since the discovery of fibroblast growth factor 21 in 2005, because of its extensive role in regulating energy metabolism in the body, it has aroused widespread interest of research teams at home and abroad. Different from other fibroblast factor families, FGF21 can be secreted into the blood and acts on various systems throughout the body. Human FGF21 is composed of 187 amino acids, mainly secreted by the liver, which regulates glucose and fat metabolism. In addition, type 2 diabetes mellitus, coronary heart disease, liver dysfunction and other diseases can lead to a slight increase in serum levels of FGF21. Recent studies have found that in mice with mitochondrial disease and in patients with mitochondrial disease, serum levels of FGF21 water Although the role of fibroblast growth factor 21 in many pathological processes has been reported and studied, their role in mitochondrial diseases has not been elucidated. In addition, few studies have been done on how fibroblast growth factor 21 regulates the function of cellular mitochondria. It can lead to abnormal metabolism of sugar, fatty acids and amino acids, not only abnormal blood lactic acid, pyruvate and alanine, but also the increase of metabolic intermediates in urine, such as 2-oxoglutarate, succinate, fumarate, malate, etc. in the case of abnormal mitochondrial function, the body through positive and negative feedback The regulation mechanism of energy metabolism is called mitochondrial energy metabolism remodeling. The energy metabolism remodeling caused by mitochondrial dysfunction mainly depends on the mitochondrial function. Cellular ATP, acetyl-CoA, NAD+, and SAM levels directly affect DNA methylation, histone acetylation, and protein phosphorylation and acetylation, and through these factors re-regulate the energy metabolism process of cells. Quantitative compensatory regulation mechanisms have been studied in the following aspects: Part I: Mechanism of energy metabolism remodeling mediated by FGF21 in mitochondrial diseases to compensate for mitochondrial dysfunction To investigate whether FGF21 is elevated in the muscle samples of patients with mitochondrial disease, to clarify the effect of FGF21 on energy metabolism, and to explore the mechanism of complex and changeable relationship between mitochondrial genotype and clinical manifestation. Materials and Methods 1) To detect and analyze the muscle groups of patients with mitochondrial disease by SYBR GREEN real-time quantitative PCR. Expression of fibroblast growth factor 21 mRNA in tissues and expression of fibroblast growth factor 21 protein in mitochondrial disease muscle tissues were analyzed by protein immunoelectrophoresis. 2) Myoblast mitochondrial dysfunction induced by mitochondrial respiratory chain inhibitors such as rotenone, oligomycin and uncoupling agent FCCP was detected by SYBR GREEN real-time quantitative PCR before and after induction of fibroblast growth factor 21. Myoblast mitochondrial function was assessed by mtDNA quantity, intracellular ATP content, citrate synthase activity and mitochondrial ROS level. 5) Oxygen consumption and extracellular acidification were measured by hippocampal energy analyzer to obtain basal cell respiration rate, ATP production and proton leakage. Maximum respiration rate, basal glycolysis rate and maximal glycolysis ability were measured by analyzing maximal respiration rate of myoblasts. 7) Protein immunohistochemical analysis of the changes of signal pathways such as mTOR-PGC1alpha in myoblasts after treatment with FGF21. 8) Rapamycin inhibited the activity of mTOR protein kinase, LY294002 Inhibiting PI3K-Akt pathway.9) shRNA reduced the expression of YY1 and PGC1alpha transcription factors and clarified the molecular mechanism of their role in promoting energy metabolism by FGF21. Results The expression of FGF21 in muscle tissue of patients with mitochondrial encephalomyopathy was significantly increased, and mitochondrial injury induced by mitochondrial respiratory chain inhibitors could up-regulate the expression of FGF21 in muscle cells. Factor FGF21 can promote the expression and translation of mitochondrial metabolism-related genes (CPT1A, CPT2, PPARy, IDH3A, GLUT1) in myoblasts, increase the number of mtDNA, enhance the activity of mitochondrial citrate synthase, increase the content of ATP, increase the oxygen consumption rate of myoblasts and promote the glycolysis of myoblasts. Cellular energy metabolism is achieved by activating the mTOR-YY-1-PGC1alpha signaling pathway in cells. Reducing the expression of PGCl alpha by rapamycin and shRNA attenuates this stimulating effect of FGF21. A tissue with mitochondrial dysfunction can reconstruct the whole body's energy metabolism process by secreting cytokines from fibroblast growth factor 21. GF21 cytokines, on the one hand, induce ketone production in the liver; on the other hand, induce fat decomposition; these ketones and fatty acids can be absorbed and utilized by muscles, brain and other organs and tissues. Dysfunction of chemical respiration can induce the elevation of FGF21 cytokines in myocytes. Muscle cells compensate for the abnormality of mitochondrial energy metabolism by secreting FGF21 cytokines. Muscle tissue acts on the muscle tissue itself by secreting FGF21 cytokines and activates the mTOR-YY1-PGC1alpha signaling pathway to enhance mitochondria. Function and oxidative respiration efficiency, on the other hand, act on liver and adipose tissue to provide energy for muscle; (4) Muscle tissues with mitochondrial dysfunction can regulate energy metabolism by secreting factor FGF21. (2) Mitochondrial gene C15620A polymorphism affects the clinical phenotype of G11778A mutation. Mitochondrial G11778A mutation is a common pathogenic mutation in Leber's hereditary optic neuropathy (LHON), but G11778A mutation is rarely reported to cause Leigh syndrome (LS). We report the clinical and pathological characteristics of a LS patient with mitochondrial G11778A mutation and investigate the relationship between the mitochondrial gene polymorphism site C15620A and the pathogenic mutation G111. Materials and Methods A 3-year-old Han Chinese child with clinical manifestations of LS was collected for related medical history, physical examination and other auxiliary examinations. Blood, cerebrospinal fluid lactic acid, uric acid, serum lipid acyl carnitine and optic nerve examination were improved. Muscle biopsy and mitochondrial gene full-length sequence analysis were performed to identify the mutation site. Spot. Cultured skin fibroblasts were isolated and cultured, and their mitochondrial respiratory chain enzyme activity, mitochondrial membrane potential and cell ATP content were analyzed to clarify the mitochondrial function of the children. The results showed that the development of the children was slower than that of the children of the same age, walking at one year and eight months, talking at two years and ten months old. Intentional tremor, right intraocular strabismus, and instability of walking began to appear. Neurological examination revealed right intraocular strabismus, walking instability and ataxia. Fundus examination showed no obvious abnormalities. Craniocerebral MRI showed symmetrical long T1 and T2 signals in bilateral medulla oblongata, pons, midbrain and cerebellum. FLARI showed high signal. Fasting blood lactate 6.0 mmol/L (normal value 1.55 mmol/L). Cerebrospinal fluid lactate 3.lmmol/L.Muscle biopsy enzyme histochemical staining revealed an increased activity of succinate dehydrogenase in some myofibrils.Mitochondrial gene analysis revealed that G11778A was a pathogenic mutation with a mutation rate of 91.6% in muscle tissue and 57% in peripheral blood. Both mutations were found in the peripheral blood of the mothers of the children, 57% and 79% respectively. Meanwhile, the activity of respiratory chain complex I, complex III and mitochondrial membrane potential of the skin fibroblasts decreased by 67%, 15% and 50% respectively. Mitochondrial gene GI1778A mutation may cause Leigh syndrome. Mitochondrial gene C15620A polymorphism may affect the clinical phenotype of pathogenic GI1778A mutation by decreasing the activity of complex III enzyme. Part III Twinkle gene expression is Leigh syndrome. Objective To summarize the characteristics of muscle pathology, gene and clinical manifestations in patients with familial extraocular muscle paralysis caused by mutation of Twinkle gene, and to analyze the pathological and clinical features of extraocular muscle paralysis caused by mutation of Twinkle gene. Two families with familial extraocular muscular paralysis were studied for clinical and other auxiliary examinations. Blood lactate, serum lipid carnitine and urine organic acid were improved. Muscle biopsy pathological findings were summarized. Twinkle gene sequencing was used to identify the pathogenic site. The results showed that the clinical manifestations of these two families were relatively mild, with only mild to moderate blepharoptosis, mild extraocular muscle palsy and progressive myasthenia. Multiple deletion mutations were found in the muscles of the probands of the two families. Conclusion Twinkle gene is a common pathogenic gene of familial extraocular muscle paralysis. Mutations of P. A475P and P. R354P are common pathogenic mutations. The clinical manifestations caused by these mutations are relatively mild. The main pathological feature of meat is a small amount of fragmented red fibers (RRFs) and COX-deficient fibers.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R746

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